Nuclear pore complexes (NPCs) cary out signal-mediated transport of proteins and RNAs between nucleus and cytoplasm, a process that is fundamentally important for control of gene expression and cell metabolism. At the present time, nuclear transport mechanisms are just beginning to come under molecular scrutiny. Developing a detailed understanding of these processes is likely to provide the potential for new therapeutic approaches to a wide spectrum of human diseases caused by microbial pathogens and genetic disorders. The long term goal of this project is to understand mechanisms of nuclear protein import at a biochemical and molecular level. This project period will involve two NPC proteins, Tpr and the p62 complex, which are linked to ligand docking at the NPC and the central gated channel, respectively. The studies also will concern several cytosolic factors required for nuclear import, including the GTPase Ran, which may be a key regulator of nuclear transport, and cytosolic factors that bind to O-linked NPC glycoproteins, including the p62 binding factor NTF2. High resolution EM analysis will be carried out on Tpr and the p62 complex, and the 3 uncharacterized subunits of the p62 complex will be molecularly cloned. Binding partners in the NPC for Ran and p62 will be identified and molecularly characterized, as will cytosolic transport factors that interact with Tpr and O-linked NPC glycoproteins. With a combination of antibodies, binding competitors and dominant negative mutants, the functions of this array of components in nuclear protein import will be examined using an in vitro assay for nuclear import involving permeabilized cells, and in vivo cultured cell systems manipulated by microinjection. Furthermore, the locations of these proteins and their associated transport steps within the 3-D structure of the NPC will be analyzed. Together, these investigations should substantially expand understanding of the series of steps responsible for signal-mediated transport through the NPC.